PROJECT SUMMARY/ABSTRACT Herpes Simplex Viruses type 1 (HSV-1) is important human pathogen that can cause a wide range of pathologies ranging from the common cold sore to disseminated end-organ disease. HSV-1 poses a significant public health due to the rise of acyclovir resistance, particularly within the ever-increasing immunocompromised patient population. It is therefore of great interest to develop new potential therapies against these viruses. Initiation, or the unwinding of dsDNA at the origin of replication, is the first step required for viral DNA synthesis. Initiation is therefore a rate-limiting step during HSV-1 DNA replication and may be a good target for the development of new antivirals. Two viral proteins are known to be essential for initiation: the origin binding protein UL9 and the single-stranded DNA binding protein ICP8. Despite the importance of this process, we still lack a detailed understanding of the molecular mechanisms and protein-protein interactions that drive initiation. In this proposal, we aim to examine how protein-protein interactions between ICP8 and UL9 contribute to initiation by mapping the residues on both ICP8 and UL9 essential for this interaction and by examining their importance in the context of infection. The C-terminal 27 amino acids of UL9 are essential for ICP8 interaction and for the initiation of origin-dependent DNA synthesis. Within this region, there is a conserved stretch of amino acids that form a linear motif (VNF, a.a. 846-848) that has been suggested to be essential for ICP8-UL9 interaction and recruitment of ICP8 to the origin. Aim 1 will test the hypothesis that this VNF motif is essential for origin unwinding and origin-dependent DNA synthesis in the context of infection. Although the region of UL9 important ICP8-UL9 is known, the reciprocal site on ICP8 that is essential for this interaction has not yet been identified. Interestingly the VNF motif that is thought to be important for UL9-ICP8 interaction has sequence similarity to a linear motif (FNF, a.a. 1142-1144) in the C- terminus of ICP8 that is important for ICP8 self-interaction. Our lab recently published data to support the notion that the FNF motif mediates ICP8 self-interaction by docking onto a conserved hydrophobic pocket on a neighboring molecule of ICP8. We hypothesize that the VNF motif in the C-terminus of UL9 may dock into the same conserved hydrophobic pocket on ICP8. We have recently shown that the C-terminus of UL9 fails to bind to a mutant version of ICP8 in which this hydrophobic pocket has been disrupted. Aim 2 of this proposal will build on these preliminary data. Knowledge gained from the experiments in this proposal will not only inform our model as to how initiation occurs, but may also pave the way for the development of new antiviral compounds that can target and disrupt this essential step during HSV-1 infection. This work will allow me to take advantage of training opportunities and mentorship to advance my career as a future physician- scientist.